Mathilde E. Boon

Prognostic indicators in breast cancer-morphometric methods

Morphometric methods were applied to predict the clinical course of individual patients with breast cancer. Measurement of tumour diameter, assessment of mitotic and cellular indices, and quantitative microscopy of nuclear features were assessed together with nuclear features and histological grades. Of the tumours from 78 patients investigated, 42 had died from metastases within 6.5 years (‘non‐survivors’), while the other 36 were alive and well without evidence of metastases at the end of the follow‐up period (minimum 6.5 years) (‘survivors’). If the tumours of the 42 non‐survivors are compared with those of 36 survivors, there are many reproducible significant differences, the most important being cellularity index and mitotic activity index, followed by quantitative microscopical nuclear parameters and nuclear and histological grade. Discriminant analysis, of the quantitative microscopical data alone showed 82% of all patients to be correctly classified as survivor or non‐survivor. By contrast with the axillary lymph node invasion status alone, or the tumour diameter and axillary lymph node status together, 59% and 64% of the patients were predicted correctly as survivor or non‐survivor. With a more realistic statistical approach of discriminant analysis, 78% of the patients were classified correctly with quantitative microscopy, in place of 54% with the axillary lymph node status, 56% with the TNM‐system and 64% with a combination of TNM system and nuclear and histological grade. Morphometry thus seems possible to predict the outcome of individual patients more accurately than with the usual staging/grading methods. This technique might therefore prove to be useful in the selection of patients for adjuvant chemotherapy.

Microwave‐stimulated diffusion for fast processing of tissue: reduced dehydrating, clearing, and impregnating times

In this paper we describe a method of preparing tissue blocks for paraffin sections within 30 min. The method is based on microwave‐stimulated diffusion reducing the dehydrating, clearing, and impregnating times by a factor of 48. The developed technique was inspired by the experimentally observed sizable temperature‐dependence of viscosity and other transport properties of liquids. It is clear that, by considering the theoretical aspects of diffusion and by analysis of the influence of the used chemicals in different tissue depths, histotechnical results can be optimized. The histotechnical microwave results are light‐microscopically excellent and indistinguishable from those of the well‐performed ‘classical’ method. The nuclear size of several cell types hardly differs in both methods. The new method is valuable in particular for individual cases in which a fast diagnosis is asked for, and in which a frozen‐section diagnosis is thought to be too unreliable. In addition, this method can be used in small research laboratories processing small quantities of histological material. The only equipment needed to prepare tissue blocks of optimal quality is the microwave oven.

Consequences of neural network technology for cervical screening: increase in diagnostic consistency and positive scores.

Screening programs for the early detection of cervical carcinoma are criticized because of the problem of false‐negative diagnoses. A successful approach for solving this problem is applying neural network technology (PAPNET®) to assist the cytotechnologist (CT) in finding the (few) abnormal cells in the smear.

The prognostic significance of morphometry in T1 bladder tumours

Only 3% of patients with T1 bladder tumours die of bladder carcinoma within 5 years (Williams, Hammonds & Saunders 1977), therefore these patients are initially treated conservatively. There would however be benefits from being able to predict which patients should be treated more aggressively. Morphometry was applied to quantitate characteristic microscopical features of the removed T1 tumours in 16 patients who had survived for five years and in seven patients dead from the tumour, in order to evaluate the prognostic value of this method. The measurements of nuclear and cytoplasmic areas were made on routine H & E sections with a graphic tablet (ASM, Leitz). Statistical analysis of the obtained data revealed that there were significant differences between the two groups. It indicated that morphometric parameters of T1 bladder tumours can have prognostic as well as therapeutic significance which will be further tested in a prospective study.

PENILE STUDIES AND HETEROSEXUAL PARTNERS - PENISCOPY, CYTOLOGY, HISTOLOGY, AND IMMUNOCYTOCHEMISTRY

The male partners of 20 women with human papilloma virus (HPV) infection (subtypes 6, 11, 16, and 18) were studied. These men were unsuspecting and allowed investigation “for scientific reasons” in the context of the abnormal findings in their female partners. Peniscopy (study of the penis with a colposcope) was abnormal in 17 cases. In the cytologic‐brush preparations of the urethra, hyperkeratosis (visualized with the modified Papanicolaou technique) occurred in all cases, but koilocytosis in only one. Both urothelial cells and squamous cells in the urethral samples were positive for HPV, as shown in the immunocytochemistry. In the histologic sections taken from flat lesions there was hyperkeratosis, dyskeratosis, and abnormal nuclear maturation. The authors concluded that, using simple techniques, subtle disturbances in maturation of nuclei and cytoplasm can be established in penile epithelium of these sexual partners. Similar changes are established in other HPV lesions. It seems likely that a significant proportion of the male population is infected in countries were HPV is an important factor in cervical carcinogenesis.

KOILOCYTOTIC LESIONS OF THE CERVIX - THE INTERRELATION OF MORPHOMETRIC FEATURES, THE PRESENCE OF PAPILLOMA-VIRUS ANTIGENS, AND THE DEGREE OF KOILOCYTOSIS

We studied the interrelation of morphometric features, the presence of human papilloma virus antigens (localized by the immunoperoxidase method), and the degree of koilocytosis in koilocytotic cervical intraepithelial neoplasia. We determined the morphometric features of the cells from the deep, the middle, and the superficial layers of the affected koilocytotic epithelium and in non‐koilocytotic immature metaplasia and squamous cervical epithelium. This approach allows quantification of disturbances of maturation in cervical epithelium. Our quantitative findings support the contention of other authors that human papilloma virus infection is associated with a morphologically distinct lesion, which forms a morphological continuum with neoplasia. It can be argued that, in addition to the degree of koilocytosis, nuclear enlargement and excessive cellular enlargement in the middle layer of the affected epithelium are viral‐related effects. With increasing immaturity of the cervical intraepithelial neoplasia all investigated viral‐related changes are less prominent. These findings suggest that in neoplastic transformation the morphological and antigen expression of human papilloma virus infection is suppressed.

MICROWAVE-STIMULATED STAINING OF PLASTIC EMBEDDED BONE-MARROW SECTIONS WITH THE ROMANOWSKY-GIEMSA STAIN - IMPROVED STAINING PATTERNS

Staining plastic sections with the Romanowsky-Giemsa method is both time-consuming and difficult. This paper reports how the staining time can be reduced to 25 min using microwave irradiation of the staining solution. It is shown that staining results depend on the fixative used, staining temperature, dye concentration and pH of the staining solution as well as on several parameters of the microwave irradiation technique. The staining patterns are improved when compared with those obtained by conventional staining of plastic sections. The colors are more brilliant and greater contrasts are observed. Basophilia, polychromasia, and orthochromasia accompanying red cell maturation are more pronounced. For white cell maturation the initial appearance of specific granules (neutrophil, basophil, and eosinophil) is more evident. Thus, cell classification is easily accomplished using the described technique. It is suggested that microwave-stimulated staining be considered for routine use.

The association between precancerous‐columnar and squamous lesions of the cervix: a morphometric study

A histological study of 208 cases found a strong association between the occurrence of precancerous cervical columnar and squamous lesions. This was not, however, directly related to cellular abnormalities. The criteria for adenocarcinoma in situ (AIS) and its precursor lesions were defined morphometrically. With increasing abnormalities of the columnar cells, changes of the glandular and epithelial architecture were more frequently observed. Of the 20 cases morphometrically classified as AIS, only four were initially diagnosed as such, of which three were cases of ‘pure’ AIS lacking squamous cell abnormalities. Quantifying abnormalities of columnar epithelium is an important aid in the identification and classification of AIS.

Pretreatment in a High-pressure Microwave Processor for MIB-1 Immunostaining of Cytological Smears and Paraffin Tissue Sections to Visualize the Various Phases of the Mitotic Cycle

In many pathology laboratories, both microwave ovens and pressure cookers are used for pretreatment of cytologic smears and paraffin sections to allow MIB-1 staining. For both methods there are two problems. First, the results cannot be used for quantitation because standardization is impossible. Second, the staining results are often suboptimal, resulting in negative staining of cells in the G1- and S-phases. When pretreatment is performed in a microwave processor, allowing microwave heating under pressure, precise temperature monitoring becomes possible. In addition, the importance of the pH of the buffer was studied using a test battery series. Optimal staining is achieved at a temperature of 115C, 10 min, pH 6. This method proved to be highly reproducible. Because the immunostaining results are optimal, the various phases of the cell cycle can be defined in the sections and smears. In addition, the perinucleolar staining of the late G1-phase is optimally visualized and nuclei of the stable pKi-67 pathway can be identified. Under suboptimal conditions, in particular, the number of cells in the late G1-phase are underestimated in the MIB-1 counts. (J Histochem Cytochem 47:1015–1020, 1999)

Human papillomavirus (HPV)‐associated male and female genital carcinomas in a hindu population. The male as vector and victim

Epidemiologic, histologic and immunohistochemical data concerning male (penile) and female (cervical and vulvar/vaginal) genital carcinomas in a Hindu population are reported. The data are from Bali, an Indonesian Hindu island in a country with a predominantly Muslim population. In contrast to the surrounding Muslim population, circumcision is rare in the people of Bali, and the rate of phimosis in grown men is very high. The Balinese epidemiologic data of 1985 to 1986 were compared with 1986 data from The Netherlands. In Bali, cervical carcinoma was the most frequent carcinoma in women, and vulvar/vaginal carcinoma ranked seven. These carcinomas were much less frequent in The Netherlands. In Balinese men, penile carcinoma is the second most frequent carcinoma, whereas it is rarely diagnosed in The Netherlands. Penile carcinomas are found in Bali in younger age groups than in The Netherlands. Based on the immunohistochemical staining results, it is estimated that over 75% of the studied Balinese genital carcinomas contain human papillomavirus (HPV). The data presented in this paper may indicate that the cofactor of impeded postcoital hygiene can be of great importance for male and female HPV‐associated genital carcinogenesis. It is clear that Balinese men, in particular men with extreme phimosis, are both vectors and victims of HPV. In The Netherlands the man is exclusively the vector of HPV.